Thesis (M. Phil.)--University of Hong Kong, 2006. / Also available online.

Orientador: Wellerson Rodrigo Scarano / Resumo: A fertilidade masculina e a espermatogênese estão diretamente ligadas à capacidade das células de Sertoli em produzir fatores associados ao desenvolvimento das células germinativas. As células de Sertoli expressam receptores para FSH e testosterona e são os principais reguladores da espermatogênese. Aproximadamente 60-70% dos casos de infertilidade masculina são considerados idiopáticos, devido aos mecanismos moleculares envolvidos na espermatogênese ainda serem desconhecidos. Estudos recentes relatam que os microRNAs (miRNAs), são capazes de modular a função testicular durante a espermatogênese e sua expressão alterada pode estar envolvida na infertilidade masculina. miRNAs podem desempenhar papel importante na resposta aos xenobióticos que têm todas as consequências adversas para a saúde. Um grupo importante de compostos orgânicos com potencial tóxico são as dioxinas, como o 2,3,7,8-tetraclorodibenzo-p-dioxina (TCDD). Modelos experimentais de exposição ao TCDD, em camundongos, demonstraram que sua exposição provoca baixa contagem de espermatozóides e atraso na puberdade. Neste estudo, analisamos o efeito do TCDD nas células de Sertoli humanas in vitro após 72h a uma dose de 10nM. Nossos resultados mostraram que as enzimas antioxidantes catalase, superóxido dismutase e glutationa peroxidase diminuíram sua atividade e confirmaram o estresse oxidativo causado pelo TCDD nesse tipo celular. 78 miRNAs apresentaram expressão alterada, com regulação positiva de 73 e regulação negat... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Male fertility and spermatogenesis are directly linked to the ability of Sertoli cells to produce factors associated with the development of germ cells. Sertoli cells express receptors for FSH and testosterone, and are the major regulators of spermatogenesis. Approximately 60-70% of male infertility cases are considered idiopathic, due to the molecular mechanisms involved in spermatogenesis are still unknown. Recent studies report that microRNAs (miRNAs) are capable of modulating spermatogenesis in testicular function and its altered expression may be involved in male infertility. miRNAs may play a role in response to xenobiotics that have all the adverse consequences for health. An important group of organic compounds that are potentially toxic are the dioxins such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Experimental models of exposure to TCDD in mice showed that its exposure causes low sperm count and delayed puberty. In this study, we analyzed the effect of TCDD on human Sertoli cells after a exposure of 72h in vitro at a dose of 10nM. Our results showed that the antioxidant enzymes catalase, superoxide dismutase and glutathione peroxidase decreased their activity and confirmed the oxidative stress caused by TCDD in this cell type. 78 miRNAs showed altered expression with upregulation of 73 miRNAs and downregulation of 5 miRNAs compared to the control group. Regarding the gene expression profile, 51 genes showed deregulated, of which 46 genes with upregulation and d... (Complete abstract click electronic access below) / Doutor

The mammalian testis is divided into two distinct compartments which carry out its principal functions. Spermatogenesis occurs within the seminiferous tubules and androgen biosynthesis primarily occurs in the interstitial space. Both these processes are entirely dependent upon the two major testicular somatic cell populations - the Sertoli and Leydig cells respectively. In human males, testicular spermatogenic and endocrine function declines during the ageing process. Of particular significance is the reported age-related decrease in Leydig cell androgen production as androgens have been suggested to play a crucial role in supporting lifelong general health in men, with low circulating testosterone linked to an increased risk of developing chronic age-related cardiometabolic diseases. However, the relationship between ageing, testicular function and disease is not fully understood, impeding the development of novel therapeutic strategies to treat age-related testicular dysfunction. In one set of studies undertaken herein, a series of novel mouse models of premature ageing were utilised to begin to dissect the process of age-related testicular degeneration. Firstly, a novel knockout-first conditional allele of a previously reported premature-ageing model driven by Cisd2 (CDGSH Iron Sulphur Domain 2) deficiency was validated and the testicular phenotype characterised and compared to that of naturally aged mice at 18-months of age. Histological analyses revealed premature testicular atrophy at 6-months of age in CISD2 deficient mice, consistent with observations of the naturally aged testis. Circulating testosterone was significantly lower in CISD2-deficient mice compared to wild-type controls at 6-months of age and the luteinising hormone/testosterone ratio was significantly elevated, indicative of compensated Leydig cell failure. mRNA expression of key genes involved in androgen production were also significantly reduced in the CISD2-deficient testis, pointing to Leydig cell dysfunction in this model of premature aging. Next, Cre/LoxP technology was used to delete Cisd2 from specific testicular cell populations to determine which cell types control/support Leydig cell function during the ageing process. Testosterone production was unaffected when Cisd2 was disrupted in either the Leydig cell population or Sertoli cell population. These observations suggest that disruption to the testicular microenvironment in which Leydig cells reside, rather than intrinsic Leydig cell ageing, may play a significant role in age-associated Leydig cell dysfunction. A second set of studies were carried out to investigate the role of leukemia inhibitory factor (LIF) signalling in the maintenance of testicular function. LIF is a pleiotropic cytokine belonging to the interleukin-6 family. In the rodent testis, LIF is expressed in fetal life and adulthood; the peritubular myoid cells thought to be the main site of production. Given their anatomical location within the testis, LIF produced by peritubular myoid cells may act on both intratubular and interstitial cells to influence spermatogenesis and steroidogenesis respectively. Indeed, LIFR is expressed in germ cells, Sertoli cells, Leydig cells as well as testicular macrophages suggesting that LIF may be a key paracrine regulator of testicular function. However, the precise role of LIF/LIFR signalling in the testis is largely unknown. As such, models of testicular cell-specific Lifr deletion were generated using Cre/LoxP technology. Analysis of these novel models of conditional LIFR ablation revealed that LIFR is dispensable in germ cells for normal spermatogenesis. However, LIFR ablation from Sertoli cells resulted in a progressive degenerative phenotype, characterised by abnormal germ cell loss, sperm stasis, seminiferous tubule distention and subsequent atrophy of the seminiferous tubules. In a final set of studies, a rat model of Leydig cell ablation-regeneration was used to determine the regenerative capacity of human adipose-derived perivascular stem cells (hAd-PSC) as a potential therapy for testicular dysfunction. Following ethane dimethanesulphonate (EDS) mediated Leydig cell ablation, primary hAd-PSCs, cultured with or without LH, IGF-1, PDGFBB, T3 and ITS supplement, were transplanted into the rat testis and Leydig cell regeneration was monitored via serial measurements of circulating luteinising hormone (LH) and testosterone. Overall, hAd- PSCs had no impact on the recovery of circulating testosterone levels. However, when pre-cultured with the cocktail of hormone/growth factor supplements, the LH spike induced by the removal of testosterone negative feedback was dampened, suggesting the transplanted cells may promote Leydig cell regeneration. Whether these cells differentiate into Leydig cells, or simply provide paracrine support to the regenerating Leydig cells remains to be determined. Although Ad-PSCs may enhance regeneration kinetics, the transplanted cells were undetectable in the testis 5 weeks post transplantation suggesting they may not survive in the context of long term xenogeneic transplantation.

Cardiac failure is one of the leading cause of deaths worldwide. Potential therapeutic approach, which overcome invasive organ transplantation and delivery of immunosuppressive drugs, is lacking nowadays. However, research of mesenchymal stem cells (MSCs) therapy displays immunomodulation potential, which can further promote variety of organ regeneration without need of drug treatment. Xenopus tropicalis immature Sertoli cells (XtiSCs) culture was established in our laboratory from juvenile Xenopus tropicalis male. XtiSCs possess immunomodulatory capacity and differentiation to cardiomyocytes after the treatment with the inhibitor of glycogen synthase kinase-3 (GSK-3) CHIR99021. To test the survival rate of transplanted XtiSCs we firstly microinjected treated cells directly inside tadpole's heart. XtiSCs proliferated there for the whole tested time period (30 days). However, after direct heart XtiSCs injection and subsequent cardiac injury in adult frog, no cells were localized in wound area. Thus, we focused on remote control of cardiac regeneration using XtiSCs without CHIR99021 treatment. We injected cells inside skeletal muscle bed and confirmed their survival and proliferation. Moreover, if cells were transplanted 3 days before heart injury, it resulted in significant reduction of fibronectin...

Dissertation (Ph.D. )--University of South Florida, 2006. / Includes vita. Includes bibliographical references. Also available online.

Sertoli cells represent the only somatic cell type within the seminiferous tubules with direct contact to germ cells. Sertoli cells significantly contribute to the development of the testicular niche in a male embryo. Their role during postnatal life is in the regulation and nutrition of germ cells and the formation of the blood-testis barrier to protect these cells. In our laboratory, we have been successful in establishing a cell line of X. tropicalis immature Sertoli cells (XtiSCs) derived from juvenile testes of X. tropicalis. The objective of this thesis was to induce the differentiation process of XtiSCs into mature Sertoli cells. In vitro experiments using several factors or primary culture from adult male X. tropicalis did not show any mature markers in differentiated XtiSCs. Another experiment using cell culture derived from pubertal mice was partially successful in the induction of the differentiation process. These results indicate that XtiSCs do have some differentiation potential into mature Sertoli cells. Part of this work was to test the ability of testicular cells isolated from juvenile males of X. tropicalis to form de novo organoids. In vitro experiments were successful when these cells were cultured in a three-layer matrigel.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Anatomy / Master / Master of Philosophy

>Magister Scientiae - MSc / Introduction The blood-testis barrier (BTB) is formed by tight junctions between adjacent Sertoli cells. The barrier formed by these tight junction helps to create a specialized environment for spermatogenesis and provide an immunological barrier to protect developing germ cells. Methamphetamine (Meth) is known as neurotoxin however, its effects on the male reproductive system, especially on Sertoli cells and, the BTB are not well established. Therefore, this study aimed to determine the effects of Meth on the TM4 mouse testis Sertoli cell line and on the integrity of the BTB permeability. Materials and Methods This study investigated the effect of selected concentrations of Meth (0.1 μM, 1 μM, 10 μM, 20 μM and 100 μM) on TM4 mouse testis Sertoli cell line for 24 until 96 hours, using two treatments: an “acute” study (24 hrs exposure) and a “chronic” study, where treatment occurred on a daily basis over 96 hrs. The following parameters were investigated: viability, cell proliferation, mitochondrial activity, monolayer permeability.